A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol, aflatoxins and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. TGF-? has been implicated as a critical promoter of genomic stability and tumor suppression; Yet, the framework by which this occurs is not known. We have observed that loss of TGF-? members ?2SP and Smad3 leads to accumulation of spontaneous DNA damage, alcohol induced liver injury and cancers including those of the liver (HCC). Cell lines, and mouse models (human and mouse) mutant for TGF-?/?2SP accumulate DNA damage that is exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Exome and genomic sequence analysis identified a somatic ?2SP mutation in alcohol associated HCC, that results in a functional disruption of TGF-? signaling. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Our hypothesis is that TGF-? is a crucial enforcer of genomic stability, and that ?2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the Fanconi pathway. Recent studies have also shown the key role of the Fanconi anemia DNA repair pathway in counteracting alcohol and acetaldehyde induced genotoxicity in mice. Importantly, the ?2SP mouse mutants phenocopy a fetal alcohol syndrome in a large number of cases and the ?2SP mutants are highly susceptible to alcohol injury. Therefore our secondary hypothesis is that TGF-?/?2SP/Smad3 are crucial for protection against aldehyde genotoxicity. Inactivation of the TGF-? pathway results in alcohol toxicity, cirrhosis and HCC. To obtain further insights into the role f ?2SP/Smad3 in DNA repair, alcohol/reactive aldehyde toxicity, and neoplasia, we propose the following: AIM 1. Determine the role of ?2SP and Smad3 in the DNA damage response, and whether ?2SP and ?2SP/Smad3 loss plays a causal role in downregulation of the Fanc/Brca pathway, impairment of the Fanc/Brca DNA damage response and interstrand cross link repair. AIM 2. Examine whether mouse mutants ?2SP, ?2SP/Smad3 and Smad3 crossed with Aldh2 null mutant mice result in a progeny that show (a) enhanced susceptibility to spontaneous tumor development as well as ethanol teratogenicity (b) whether the hepatocytes and MEFs derived from such crosses undergo progressive DNA damage. Potentially these studies will generate a robust model for alcohol induced liver toxicity. AIM 3. (a) Determine whether the TGF-? pathway and DNA repair genes Fanc/Brca are prognostic factors in cirrhosis progressing to HCC, and (b) Identify novel genetic loci that predispose to HCC. c) Expand to fine mapping. Significantly, studies proposed under this application should provide the scientific community with a new understanding of the instrumental regulators in alcohol induced liver injury and neoplasia.